The overall objective of the research is to identify, characterize and isolate the cellular events and components involved in the intestinal transport of bile acids. To accomplish this objective, there are two inter-related specific aims. The first is to identify the intracellular pathway of bile acid transport. For these studies, isolated ileal cells are exposed to UV light (photolysis) in the presence of a radiolabeled photolabile bile acid derivative. The photolyzed cells are subjected to subcellular fractionation, using differential centrifugation and sorbitol gradient separation, followed by SDS-PAGE. The total amount of radioactivity and specific radioactivity, as well as the amount of radioactivity incorporated into bile acid-binding polypeptides, are calculated for each subcellular fraction. Studies will determine the intracellular pathway with respect to short times of incubation using flash photolysis. Increasing concentrations of photolabile bile acid derivative and different ratios of derivative to natural bile acid will explore the possibility that association with bile acid-binding polypeptides is dependent on intracellular bile acid concentration. Bile acid-binding polypeptides will be characterized using immunoprecipitation, detergent phase separation and single and two-dimensional gel electrophoresis. The second aim is to isolate bile acid transporter proteins. For these studies, enriched subcellular fractions are solubilized and subjected to protein purification using a glycocholate derivative - Sepharose- 4B affinity column. The purified protein, as assessed by SDS-PAGE, is established as a transporter protein by: a) demonstration of characteristics of the natural bile acid transport system following incorporation of purified protein into phospholipid liposomes and b) specific inhibition of bile acid uptake into membrane vesicles by antiserum directed against purified protein. Antiserum also will be used to analyze the immunoidentity of bile acid-binding polypeptides in other subcellular fractions or similar subcellular fractions from different tissues. These experiments should provide not only greater understanding of factors involved in bile acid transport, but also insight into the basic but unresolved question of how transepithelial translocation occurs for all transport systems.